Until now several commercially valuable proteins produced in recombinent bacteria were largely found to be recoverable primarily in a biologically inactive conformation. It has been necessary to refold those proteins into a biologically active conformation to obtain a profitable yield. By the use of recombinant DNA technology it is now possible to transfer DNA between different organisms for the purposes of expressing foreign proteins in biologically active conformation. Such transfer usually involves joining appropriate fragments of DNA to a vector molecule, which is then transformed into a recipient organism. Transformants are selected by a known marker on the vector, or by a genetic or biochemical screen to identify the cloned fragment. Vectors contain sequences that enable autonomous replication within the host cell, or allow integration into a chromosome of the host.
If the cloned DNA sequence encodes a protein, a series of events must occur to obtain synthesis of this foreign protein in a biologically active form in the host-cell. Promoter sequences must be present to allow transcription of the gene by RNA polymerase, and a ribosome binding site and initiation codon must be present in the transcribed mRNA for translation by ribosomes. These transcriptional and translational recognition sequences are usually optimized for effective binding by the host RNA polymerase and ribosomes, and by the judicious choice of vectors, it is often possible to obtain effective expression of many foreign genes in a host cell.
While many of the problems of efficient transcription and translation have been generally recognized and for the most part, overcome, the resulting synthesis of a foreign polypeptide frequently leads to low, or negligible, yields of biologically active protein. The over-expressed proteins often form inclusion bodies composed of aggregated inactive protein. The aggregated proteins must then be recovered and attempts made to refold the protein in vitro. Heterologous proteins may remain soluble in vivo, but may be subjected to proteolysis because of incorrect or incomplete folding in the unusual cellular environment (Kane et al., TIBTECH, 6, 95, (1988)). From such observations, it has become clear that factors that influence the folding of nascent polypeptide chains in vivo can have a considerable effect on the yield of foreign proteins produced from expression vectors in transformed cells.
The state of the art is illustrated by U.S. Pat. No. 5,158,875 to Miller et al. This patent to a method of producing insulin-like growth factor (IGF-1) recites a refolding step subsequent to harvesting IGF from the host cell. The step is recited as follows. "[R]efolding said Met-lys-IGF-I into its biologically active conformation." Clearly a commercially important limitation in the art is the inability to obtain commercial yield of some heterologous proteins in biologically active conformation.
A group of ancillary proteins, known as molecular chaperones, many of which are heat shock proteins (HSP), have been identified that have a profound influence on folding, recycling and synthesis of proteins. Prokaryotic molecular chaperones or HSP include such proteins as SecB, DnaK (hsp70 homologues in eukaryotic cells), DnaJ, GrpE, GroES (cpn10 homologues in eukaryotic cells) and GroEL (cpn60 or hsp60 in eukaryotic cells) (LaRossa et al., Mol. Micriobiol., 5 (3) , 529, (1991)) . The heat shock phenomenon, responsible for inducing the HSP or chaperones, was first defined as a response to a temperature up-shift. Subsequent work has Shown that exposure to a variety of stresses including phage infection, macrophage envelopment, as well as the presence of organic molecules and heavy metals can also trigger the heat shock response. Alternatively, the heat shock response may be artificially activated by the creation of various genetic mutants which constitutively express high levels of HSP, including molecular chaperones (LaRossa et al., Mol. Micriobiol., 5(3), 529, (1991)). The most extensively studied molecular chaperones are the GroES and GroEL proteins, and their eukaryotic homologues, collectively known as chaperonins.
The groE locus was first identified in studies of mutations that interfere with the assembly of the heads of bacteriophages lambda and T4 and the assembly of the tail of bacteriophage T5. The groE locus contains two genes, groEL and groES, both of which are essential for cell viability. The groEL and groES genes encode polypeptides with apparent relative molecular masses of 65,000 and 15,000, respectively, which are among the most abundant proteins in the cell. Both GroEL and GroES polypeptides are found in the cell as oligomeric structures. The GroEL protein is an ATPase, and the GroEL and GroES proteins form a complex with each other in the presence of MgATP and other nucleotides but not in their absence (Ellis et al., Biochem. Soc. Syrup., Kay et al. (Ed.) No. 55, 145, (1989)).
From in vitro studies, it is clear that a very wide range of unfolded or partially folded polypeptides interact with the GroEL protein, and that this interaction influences the subsequent folding reaction. When diluted from a denaturing solution, most proteins rapidly collapse to a non-native structure. Then, depending on the particular protein, its concentration, the temperature, and other factors, the non-native state will either progress to an active folded species, or will partition to a misfolded or aggregated state that is non-functional. If GroEL is present during dilution of unfolded proteins from the denaturing solution, the non-native proteins will bind to GroEL and form a stable binary complex. (Goloubinoff et al., Nature, 342, 884, (1989)). This has two possible consequences. For proteins that would normally continue to refold spontaneously, the folding reaction is arrested. In addition, proteins that would misfold and aggregate under these conditions are now bound to GroEL and remain in solution. Proteins bound to GroEL can be subsequently released by the addition of either ATP, or a combination of ATP and the chaperonin GroES. Under these conditions, proteins that would have folded anyway without the addition of GroEL, may continue to do so. However, proteins that would have aggregated are now directed towards the productive folding pathway. Survey-type experiments reveal that most E. coli proteins in a complex mixture, and many individual purified proteins, will interact with GroEL in this manner (Viitanen et al., Protein Sci., 1, 363, (1992); Gatenby, Plant Mol. Biol., 19, 677, (1992)).
In vivo studies also reveal that chaperones influence the successful folding of proteins into active molecules within cells. By using a cloned groE operon (encoding the genes for proteins GroES and GroEL) on a multicopy plasmid, it is possible to suppress a wide range of temperature sensitive missense mutations, indicating that successful folding of thermolabile enzymes can be restored by over-expression of the groES and groEL genes (Van Dyk et al., Nature, 342, 451, (1989)). By using combinations of compatible plasmids, where one plasmid encodes the groE operon, and the second plasmid encodes an overproduced foreign protein, it has been shown in several reports that the enhanced levels of molecular chaperones in the cell result in considerable improvements to the yield of functional recombinant protein (Goloubinoff et al., Nature, 337, 44, (1989); Berry-Lowe et al., Plant Physiol., 99, 1597, (1992)).
There are, however, several inherent problems in the use of the dual plasmid system for enhanced production of foreign proteins in bacteria, especially for scale-up operations. Cloning of the groE operon on a multicopy plasmid leads to very high levels of synthesis of the GroES and GroEL proteins, with cells often producing 30-50% of their total cellular protein as GroEL. This results in a reduced biosynthetic capacity in the cell for other proteins, including the desired foreign protein, and overall yields may be reduced compared to the possible maximum., This high level production also influences groE plasmid stability, resulting in plasmid deletions that reduce expression of the groE genes. These deletions have been observed during long-term storage of strains, and during large scale culture of cells in the 10-200 1 range (Kalbach et al., Enzyme Microbiol. Technol., (in press) (1993)). In addition, it is apparent that the proper folding of proteins requires the concerted action of a number of different molecular chaperones, and is not confined to just GroES and GroEL (Langer et al., Nature, 356, 683, (1992)). Although the folding of some proteins is facilitated in vivo by groE over-expression, others do not respond to groE (Gatenby et al., Trends in Biotechnology, 8, 354, (1990)). The over-expression of a range of molecular chaperones may be required to assist the correct folding of proteins that do not respond to groE over-expression alone. This could be achieved by cloning all of the chaperone genes on compatible plasmids. However, this method would probably result again in strains harboring large unstable plasmids. A more preferred method might involve general activation of a broad range of native heat shock proteins in conjunction with the expression of the foreign protein.
It now appears that the regulation of general expression of heat shock proteins may be controlled by a few proteins, termed sigma factors. Sigma-32 is the most studied of these factors. Grossman et al. (Cell, 38, 383, (1984)) disclose that the htpR gene of E. coli encodes a positive regulator of the heat shock response. Over-expression of the htpR gene and subsequent purification of the protein product revealed a 32kDa protein termed, Sigma-32. Grossman et al. teach that the sigma-32 protein is responsible for transcription initiation at heat shock promoters. Grossman et al. additionally suggested renaming the htpR gene rpoH, and currently the two terms are used interchangeably.
By way of confirming the general function of the Sigma-32 factor, Yano et al. (J. Bacteriol., 172, 2124, (1990)) teach a rpoH mutant that cannot grow at or above 34.degree. C. since it produces an altered sigma-32 protein that is largely deficient in the transcription of the heat shock genes. Extragenic suppressor mutations endowed the rpoH mutant strain with the ability to grow at 40.degree. C. and markedly enhanced the rate of sigma-32 synthesis and the induction of heat shock proteins.
Coupling of the regulation of the sigma-32 factor with the expression of foreign proteins is disclosed in the art. Debouck et al. (EP 216,747) teach a method of expressing a foreign polypeptide coding sequence in a htpR.sup.- E. coli mutant, not stably expressed in htpR.sup.+ E. coli strains due to degradation by proteases under the control of the htpR.sup.+ gene. It is further disclosed that the htpR.sup.- mutants do not code for wild-type sigma32 and thus are not able to undergo normal heat shock response. Similarly, Haley (WO 8503949) teaches the engineering of htpR.sup.- mutants deficient in the production of the La protease for the expression of foreign polypeptides from E. coli. Abrahamsen et al. (EP 225,860) disclose a method for the expression and secretion of proteins in Gram negative bacteria by engineering into the host cell a DNA construct encoding a foreign protein where expression of the protein is dependent on the induction of the native htpR gene. The method of Abrahamsen relies on the generalized expression of heat shock proteins to give leakage of the periplasmic membrane of the transformed host, allowing for the secretion of the foreign protein into the growth medium.
Easton et al. (Gene, 101, 291, (1991)) disclose a method for the production of bovine insulin-like growth factor 2 (bIGF2) in the cytoplasm of E. coli containing a rpoH mutation. The bIGF2 was produced in inclusion bodies and constituted 20-25% of the total cellular protein. Similarly, Obukowicz et al. (Appl. Environ. Microbiol., 58, 1511, (1992)) teach novel rpoH mutants for enhanced heterologous gene expression. Expression studies utilizing the recA or araBAD promoter and the phage T7 gene 10L ribosome-binding site showed that increased accumulation levels of a number of representative heterologous proteins were obtained in the rpoH mutants. Examples of heterologous proteins produced included human and bovine insulin-like growth factors 1 and 2, and bovine placental lactogen.
The methods recited above are useful for effecting the expression of recombinant proteins which are susceptible to sigma-32 dependent proteolysis or have a need to be secreted into the growth medium. With one exception, all the above cited art teaches that the down-regulation of the sigma-32 factor results in increased expression of foreign polypeptides. The most popular explanation for this phenomenon is that the rpoH.sup.- mutants are deficient in the sigma-32 factor which results in impaired heat shock response. Higher expression of foreign proteins is expected in these mutants due to the absence of proteases resulting from the impaired heat shock response. Although the method of Abrahamsen (EP 225,860) teaches induction of the rpoH gene and the subsequent increase of heat shock proteins the primary motivation is to allow for the secretion of the protein by altering the permeability of the periplasmic membrane and no indication is given as to the levels of foreign protein expression by the host cell. It should be noted that inherent in the method of Abrahamsen is a fully functional wild-type DnaK protein. Additionally, it is known in the art that wild-type DnaK is functional in the facilitation of native protein secretion from E. coli (Wild et al., Genes Dev., 6, 1165, (1992)).
Recent genetic evidence indicates that there are key regulator functions for another heat shock protein, DnaK, at the levels of synthesis, activity and degradation of sigma-32. DnaK, (a member of the Hsp70 protein family) is the 70kDa product of the dnaK heat shock gene and is thought to work in concert with other heat shock proteins to effect the refolding or synthesis of damaged cellular proteins (LaRossa et al., Mol. Micriobiol., 5 (3), 529, (1991)) . Mutations in dnaK, dnaJ and grpE have been shown to cause partial stabilization of sigma-32 and loss of repression of heat shock gene transcription normally found in wild-type cells after temperature downshift (Straus et al., Genes Dev., 4, 2202, (1990) and Straus et al., Genes Dev., 3, 2003, (1989)). The mechanism by which DnaK, DnaJ and GrpE regulate the activity and stability of sigma-32 is assumed to rely on their concerted activity as chaperones. This activity involves the ATP-dependant binding to substrates of DnaK and the stimulation of hydrolysis of DnaK-bound ATP by DnaJ and GrpE (Gamer et al., Cell, 69, 833, (1992)). It has been proposed that DnaK interacts with sigma-32 and dissociates it from RNA polymerase, thereby rendering it accessible to cellular proteases.
In contrast to the rpoH mutants, dnaK mutants have not been used heretofore for the expression of foreign polypeptides from recombinant bacteria. It would appear, in fact, that the presence of the DnaK protein may be necessary for the expression and secretion of some native and recombinant proteins. Wild et al. (Genes Der., 6, 1165, (1992)) teach that translocation of alkaline phosphatase, was inhibited in dnaK mutant E. coli strains suggesting that export of this protein probably involves the DnaK protein. Murby et al. (Biotechnol. Appl. Biochem., 14, 336, (1991)) disclose the coexpression of three recombinant proteins (human proinsulin, rat protein disulfide isomerase and the alpha.1.2.-chain of human T-cell receptor) as fusion proteins. Unexpectedly, the fusion proteins were found to be associated with DnaK and GroEL, indicating a function for these HSP in the expression of the foreign proteins. Similarly, Hellebust et al. (J. Bacteriol., 172, 5030, (1990)) teach that protein A expressed by a recombinant E. coli is associated with the DnaK protein. Although it is clear that DnaK functions in the regulation of the sigma-32 factor, it is equally clear that it plays an important role in the facilitation of expression of various recombinant proteins.
The present invention provides a method which relies on a dnaK mutation for the purpose of enhancing the expression of biologically active heterologous proteins. The instant method is clearly distinguished from the art. The dnaK defective strain produces a defective DnaK protein. Sigma-32 is present in higher amounts in this mutant. The present invention takes advantage of this intracellular condition to enhance expression of biologically active heterologous proteins. Clearly the art has taught that foreign polypeptide expression is enhanced by lowering levels of sigma-32, ostensibly due to the lower protease levels achieved by the impaired heat shock response. The art also teaches that the wild-type DnaK protein is necessary and useful in the expression and secretion of heterologous proteins.
The instant invention provides a means to improve the yield of active foreign proteins produced in bacteria without having to subject the culture to a temperature shift, and without the necessity of a dual plasmid expression system. It is contemplated that the production yield of commercially valuable proteins, such as hormones, enzymes and other proteins can be improved if expression is performed by the instant method.